The goal of this application is to obtain funding through the """"""""Restoration of New Investigator Pilot Projects Adversely Affected by Hurricane Sandy"""""""" opportunity. In October 2012, I had collected strong preliminary data and was preparing an R01 application for the June 2013 deadline. My research was seriously impacted by the after-effects of Super-storm Sandy. My losses include a colony of connexin43 (gap junction) mutant mice, generated as recipients for cell-transplantation studies, a breeding colony of rats, as well as precious frozen cell lysates and tissue samples awaiting molecular and histological analyses. Experiments proposed in this application will restore pilot project data towards a competitive grant submission, anticipated for summer/fall 2014. My long-term research interest is improving the therapeutic use of pluripotent stem cells (PSC) for heart diseases. PSC allow gene manipulation and can be differentiated into functional heart muscle cells in the dish. Already, PSC are being used for personalized medicine approaches, and PSC will be a suitable source for autologous cell replacement therapy in the future. To be seriously contemplated for therapeutic applications, however, several challenges remain, including long-term survival and functional engraftment of transplanted cells. Engraftment of cells is enhanced by intercellular connections, or cell-cell contacts, formed by specialized proteins. Cell contacts provide structural tissue support (mechanical junctions: adherens junctions;desmosomes) and allow fast impulse propagation and the synchronous contraction of the heart muscle (electrical junctions: gap junction channels). Indeed, transplantation of cardiomyocytes derived from PSC lacking the gap junction protein connexin43, lead to increased arrhythmogenesis in mice. Our preliminary data demonstrate that stem cell derived cardiomyocytes form very few functional gap junction contacts with native cardiomyocytes. The overarching hypothesis of this proposal is that an increase in gap junction channel formation and function will improve the therapeutic efficacy of stem cell derived cardiomyocytes. I will employ three different approaches to test this hypothesis. Based on preliminary data, I will investigate how the formin protein Daam1 enhances gap junction formation. Further, I will analyze the influence of forced gap junction channel formation for cell engraftment using PSC expressing a mutant gap junction channel, K258stop. Additionally I will use a high throughput screen to identify small molecules regulating gap junction expression in stem cell derived cardiomyocytes. While experiments proposed in this study will elucidate ways to enhance the formation of gap junctions in stem cell derived cardiomyocytes, the mechanisms identified might very well also be applicable and therapeutically relevant to the treatment of cardiac diseases related to changes in gap junction formation.
Pluripotent stem cells are currently the most suitable source for autologous cell replacement therapy and personalized medicine approaches. I hypothesize that increasing gap junction channel formation will improve the therapeutic efficacy of stem cell derived cardiomyocytes and I will regenerate pilot data lost due to hurricane Sandy towards three specific aims. While experiments proposed in this study will elucidate ways to enhance the formation of gap junctions in stem cell derived cardiomyocytes, the mechanisms identified might very well also be applicable and therapeutically relevant to the treatment of cardiac diseases related to changes in gap junction formation.
